Abstract

Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.

Overview of experimental assay. (A) Schematic representation of the experimental design. A single DNA hairpin was tethered to functionalized beads inside a fluidics chamber. One strand of the hairpin (blue) is attached through a dsDNA handle to a bead held in the laser trap, while the complementary strand (red) is attached to a bead on top of a mobile micropipette (Methods). At a constant tension (f) the strand displacement and primer extension activities of the polymerase (red triangle) are detected as a change in distance between the beads, Δx1 and Δx2, respectively. (B) Representative replication traces of the wild-type (blue) and sdd mutant (green) polymerases showing in detail the distance changes during strand displacement (s.d., Δx1) and primer extension (p.e., Δx2) activities (f ∼ 11 pN). See Fig. S3 for additional experimental traces at different tensions.

Tension and sequence dependencies of the wild-type and sdd mutant rates. Only activities that replicate the full hairpin length were considered (Fig. S4A). (A) The tension dependency of the average strand displacement rate (Vsd) of the wild-type polymerase with and without pauses (full and empty blue circles, respectively) is well explained by our active model (solid blue line). (B) During strand displacement the wild-type polymerase spends longer times (blue, f ∼ 4 pN, N = 15) at the more stable hairpin positions (dotted lines). This behavior is well predicted by the proposed model (orange). For comparison, the experimental force-unzipping curve of the hairpin is shown in gray. (C) The different tension dependencies of the average strand displacement rate (Vsd) of the mutant with and without pauses (full and empty green circles, respectively) can be explained with the same active model when pause kinetics are included in (solid green line) or excluded from (dashed green line) the model. (D) Representative position versus time traces of the mutant polymerase during strand displacement. Identified pause events are shown in red, long pauses are located at the GC rich positions of the hairpin (gray lines). Insert shows the sdd mutant velocity distribution during strand displacement conditions (Fig. S5C). (A and C) For both polymerases, full and empty red circles represent the average primer extension rates (Vpe) with and without pauses. Solid red lines represent the sequence independent velocity value used in the model (128 nt/s). Error bars represent the standard error.

DNA unwinding model. (A) Diagram showing notation used for modeling the polymerase movement during strand displacement conditions. M (red circle) defines the range of interaction between the polymerase and the junction; m is the number of ssDNA template nucleotides between the polymerase and the junction; l is the total number of replicated nucleotides and L is the full length of the hairpin. (B) Proposed DNA unwinding mechanism. Figures show the schematic representation of the Phi29 DNA polymerase with the DNA substrate during strand displacement (). The protein is diagrammed in two levels. The upper level contains the exo (green), the TPR2 (blue) and thumb (orange) domains. The rest of the protein is shown as a gray circle. (1) and (2) Template bending at the TPR2-exo tunnel and steric exclusion of the complementary strand may generate mechanical stress at the fork junction promoting the unwinding of the first 2 bp (blue) of the fork. dNTP binding and hydrolysis fueled the polymerase forward movement. (3) For the sdd mutant, mechanical stress at the junction during unwinding of the more stable fork positions (where m = 0) could lead to destabilization of the template-tunnel interactions, favoring the entrance to the Long Pause 2 state. Fork destabilization by external tension (f) favors DNA unwinding, preventing the entrance to and promoting the exit from the inactive Long Pause 2 state. In (2) and (3) the initial position of the fork is shown in light gray.